Use of Akkermansia muciniphila for treating inflammatory conditions

ABSTRACT

The invention relates to use of Akkermansia muciniphila, a mucin-degrading bacterial species found in the human gut, for treating undesirable inflammatory activity not caused by any metabolic disorder and/or obesity, especially for example undesirable airway inflammatory activity as seen with asthma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of co-pendingU.S. application Ser. No. 15/766,215 filed Apr. 5, 2018, which is a 35U.S.C. § 371 National Phase Entry Application of InternationalApplication No. PCT/GB2016/053097, filed Oct. 5, 2016, which designatesthe U.S. and which claims priority to U.S. Application No. 62/237,131,filed Oct. 5, 2015, the contents of each of which are incorporatedherein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 5, 2018 isnamed P224214US_WO_ST25_3429641.txt and is 858 bytes in size.

FIELD OF THE INVENTION

The invention relates to the use of Akkermansia muciniphila as animmunomodulatory biotherapeutic agent for treating asthma. Moregenerally, it relates to use of Akkermansia muciniphila, amucin-degrading bacterial species found in the human gut, for treatingundesirable inflammatory activity unrelated to any metabolic disorderand/or obesity. In this context, “unrelated to” will be understood tomean “not caused by”. Of especial interest is use of Akkermansiamuciniphilia in the prophylaxis and/or treatment of airway inflammationin asthmatic patients identified as having a lower level of that microbein the gut compared to healthy individuals. Commonly such asthmatics canbe expected to have asthma which can be categorized as severe.

BACKGROUND TO THE INVENTION

The human microbiome is a microbial community, which can be described asthe sum of all microbial life living in or on specific sites of thehuman body. Recent advances in DNA sequencing techniques havefacilitated more in-depth analysis of the microbiomes of the gut, skin,genito-urinary tract and the lung, revealing a microbial super organresiding symbiotically with host mucosal surfaces. It is becoming betterappreciated that the composition and activity of the microbiome hassignificant metabolic, nutritional and immunological effects on the host(1), The microbiome evolves within a host from birth, constantly beingfine-tuned to maintain a homeostatic balance with the host's immunesystem. This evolution is influenced by host factors, such as theadaptive and innate immune responses, external factors such as diet,medication and toxin exposure, and illness.

The gastrointestinal tract has the greatest number and diversity ofmicrobes, with approximately 100 trillion microbes residing in the gut(2 to 10 times the total number of human cells in the entire body),while the collective genomes of the gut microbiome contain approximately100 times more genes than the human genome. These microbes are highlyadapted to survive within complex community structures, requiringnutrients from other microbes and/or host processes. Interestingly,using sequencing approaches, over 1,000 different bacterial species havebeen identified within the gut microbiome. However, a specificindividual's microbiome typically contains only 300-500 differentspecies, leading to enormous inter-individual variability in microbiomecomposition. This variability is even more pronounced when patients withdiseases, such as inflammatory bowel disease, are compared to healthyindividuals, supporting the concept that an imbalance of certainmicrobes (i.e. dysbiosis) within the microbiome may contribute toaberrant inflammatory and metabolic responses (2), Similarly,alterations in the microbiome of the lung have been associated with lungassociated disorders such as asthma and chronic obstructive pulmonarydisease (3).

The microbiome supports the development of epithelial barrier functionand integrity, while promoting potent tolerance and protective immunemechanisms within mucosal tissues (4). Microbes have direct effects onhost immune responses and metabolites derived from microbialfermentation of nutrients within the gut not only contribute to hostenergy intake, but also significantly influence host immunologicalresponses (e.g. short-chain fatty acids and histamine) (5, 6).Appropriate cellular and molecular networks involve innate patternrecognition receptor activation, T and B cell polarization andexpansion, secretion of a wide range of effector and regulatorycytokines and host metabolites. Ultimately this trialogue between themicrobiome, immune cells and tissue cells within the gut results in theestablishment of optimal digestive capabilities, gut motility, immunetolerance to foods and certain microbial antigens, and protectionagainst pathogens.

The immunological consequences of bacterial processes within thegastrointestinal tract have effects on organs distant to the gut itself(7). For example, respiratory inflammation has been treated in murinemodels by oral administration of certain probiotic bacteria such asLactobacilli and Bifidobacteria (8, 9). In addition, a reduced gutbacterial diversity early in life increases the risk of later lifeasthma (10).

Akkermansia species are commensal microorganisms. They have beenisolated from the microbial flora within the human gastrointestinaltract (11). The immune system within the gastrointestinal tract cannothave a pronounced reaction to members of this flora, as the resultinginflammatory activity would also destroy host cells and tissue function.Therefore, some mechanism(s) exist whereby the immune system canrecognize commensal non-pathogenic members of the gastrointestinal floraas being different to pathogenic organisms.

This ensures that damage to host tissues is restricted and a defensivebarrier is still maintained. Akkermansia muciniphila has been shown in amurine model to modulate pathways involved in establishing homeostasisfor basal metabolism and immune tolerance toward commensal microbiota(12).

Akkermansia muciniphila has been previously reported to be reduced infaecal samples from obese individuals and patients with inflammatorybowel disease (13). Akkermansia muciniphila is associated with ahealthier metabolic status and better clinical outcomes after a calorierestriction intervention in overweight/obese adults (14). Indeed,published International Patent Application WO 2014/075745, and publishedInternational Application WO 2014/076246 claiming priority therefrom,concern the protective effects of Akkermansia muciniphila in obeseindividuals and propose administration of Akkermansia muciniphila toindividuals exhibiting obesity and/or metabolic disorders such as type 2diabetes to provide various beneficial effects (15). Administration ofAkkermansia muciniphilia to obese and type 2 diabetic mice has beenshown to correlate with an improved metabolic profile and to be able toprotect against high fat diet-induced metabolic disorders. Beneficialeffects were reported of interest in relation to associated gutinflammation including increase of intestinal levels of endocannabinoids(16). However such studies provide no information relevant toameliorating other forms of inflammation, including gut inflammation ofdifferent etiology.

It has now been discovered that Akkermansia muciniphila levels aredecreased in non-obese asthma patients with uncontrolled symptoms andthis bacterium is protective in non-obese animal models of respiratoryinflammation. It has been previously described that the same bacteriumexerts immunoregulatory effects within the gastrointestinal tract,possibly directly and indirectly via the digestion of intestinal mucinsand subsequent effects on SOFA secretion by the microbiome (see again12). Lower prevalence of Akkermansia muciniphila has also previouslybeen correlated with IgE-mediated atopic disease in a small number ofallergic children (17). However those studies provide no more than amere correlation and provide no foundation for extrapolation of anybeneficial effect of oral administration of the same microbe at a siteremote from the gastrointestinal (GI) tract, nor evidence of a route bywhich this might be achieved.

SUMMARY OF THE INVENTION

Studies reported herein for the first time show linkage in humansbetween decreased gastrointestinal levels of Akkermansia muciniphila andthe severity of a respiratory disease, more particularly asthma, withdemonstration of protective effects in therapeutically relevant murinemodels of respiratory inflammation. Furthermore, it has bean shown thatAkkermansia muciniphila can induce ILT4 (immunoglobulin-like transcript4) expression on human monocyte derived dendritic cells (MDDCs). ILT4(also sometimes referred to as LILRB2) is known to be important forinducing regulatory T cells which can secrete IL-10, ananti-inflammatory cytokine. Moreover, mice administered Akkermansiamuciniphila have been found to have increased regulatory T cells thatsecrete IL-10 amongst lung tissue lymphocytes. These observations, asdescribed more fully below, provide for the first time foundation forprobiotic use of Akkermansia muciniphila strains for the prophylaxisand/or treatment of respiratory inflammation, e.g. as associated withasthma, especially severe asthma, and other undesirable inflammationindependent from and unconnected with any metabolic disorder and/orobesity.

It will be understood that the metabolic disorders referred to above arerelated to a disorder of energy utilization and storage. A common signof metabolic disorder is central obesity or overweight with adiposetissue accumulation particularly around the waist and trunk. Other signsof metabolic disorder include high blood pressure, decreased fastingserum HDL cholesterol, elevated fasting serum triglyceride level (VLDLtriglyceride), impaired fasting glucose, insulin resistance, orprediabetes. Associated conditions include hyperuricemia, fatty liver(especially in concurrent obesity) progressing to nonalcoholic fattyliver disease, polycystic ovarian syndrome (in women), erectiledysfunction (in men), and acanthosis nigricans.

Severe asthma as referred to herein can be understood and defined asfollows: When a diagnosis of asthma is confirmed and comorbidities havebeen addressed, severe asthma is defined as “asthma which requirestreatment with high dose inhaled corticosteroids (ICS) plus a secondcontroller (and/or systemic CS) to prevent it from becoming“uncontrolled” or which remains “uncontrolled” despite this therapy.”(18)

In one aspect, the present invention thus provides a compositioncomprising one or more Akkermansia muciniphila strains for use in theprophylaxis and/or treatment of undesirable inflammatory activitywherein said inflammatory activity is not caused by a metabolic disorderand/or obesity.

Such a composition may be a probiotic composition for delivery of theone or more Akkermansia muciniphilia strains in the GI tract, preferablyfor oral administration, e.g. in the form of a beverage or other form ofnutritional composition for oral consumption. It may be in the form of apharmaceutical composition for administration of one of more Akkermansiamuciniphila strains in the GI tract, preferably by oral administration.The composition may be a strain-release composition which is notingested in which case said use further comprises the pre-step ofcontacting said composition with a liquid whereby the one or moreprobiotic strains are released into the liquid and the resultingprobiotic liquid composition is then ingested. It is envisaged that theprobiotic composition employed may alternatively be formulated andpackaged for aerosol delivery.

Akkermansia muciniphila strains for use in applying the invention willbe understood to include naturally-occurring strains and additionallyvariants and genetically-modified mutants of naturally-occurringAkkermansia muciniphila strains, in any form, provided that desiredimmunomodulatory effect when in the gut is maintained.

Alternatively, the invention provides a method for the prophylaxisand/or treatment of undesirable inflammatory activity which comprisesadministration of one or more Akkermansia muciniphila strains to the GItract, wherein said inflammatory activity is not caused by a metabolicdisorder and for obesity.

The invention will be further described below with reference thefollowing figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates that Akkermansia muciniphila levels decrease inuncontrolled asthma patients with increased symptom severity, while FIG.1B demonstrates that the decrease in Akkermansia muciniphila levels isunrelated to obesity. The data is represented as relative abundance ofAkkermansia muciniphilia, based on 16s rRNA gene sequencing employingfaecal samples.

FIG. 2 shows that Akkermansia muciniphila levels decrease in asthmapatients with severe disease which is unrelated to obesity relying onquantitative PCR and Akkermansia muciniphilia-specific primers toanalyse A. muciniphila genomic DNA in faecal samples from asthmapatients with varying severity of asthma. A patient was included to thesevere asthma group if he/she fulfilled one or both major and at leasttwo minor criteria as defined by Chung et al (18). Patients who did notfulfil these criteria were enrolled to mild or moderate asthma groups.The data is represented as genome copy number per ng of DNA, assessedusing quantitative PCR.

FIG. 3A is a graph showing the suppressive effect of Akkermansiamuciniphila feeding on inflammatory cell recruitment to the lungs ofhouse dust mite (HDM) extract acutely challenged mice, with FIG. 3Bhighlighting the decrease in inflammatory neutrophils.

FIG. 4A shows the suppressive effect of Akkermansia muciniphila feedingon inflammatory innate cell recruitment to the lungs during thehealing/resolution phase following chronic challenge of mice with housedust mite extract, with FIG. 4B highlighting the decrease in lymphocytesubsets. Data presented as mean+/− SEM. Data analysed by one-way ANOVAwith Tukey p value correction. Data log transformed where necessary tostabilize variance. P values of less than 0.05 were consideredsignificant.

FIG. 5A illustrates the suppressive effect of Akkermansia muciniphilafeeding on eosinophil recruitment to the lungs following re-challengewith house dust mite extract in the chronic house dust mite challengemodel, with FIGS. 5B and C highlighting the decrease in Siglec-F^(high)eosinophils in the lung tissue.

FIG. 6 illustrates the suppressive effect of Akkermansia muciniphilafeeding on IgE production, specific to house dust mite extract,following re-challenge with house dust mite extract in the chronic housedust mite challenge model.

FIG. 7A is a graph showing the suppressive effect of Akkermansiamuciniphila feeding on inflammatory cell recruitment to the lungs ofovalbumin (OVA) challenged mice, with FIG. 78 highlighting the decreasein inflammatory eosinophils.

FIGS. 8A and 8B illustrate that cells within the lung tissue ofAkkermansia muciniphila fed animals secrete significantly lessinterleukin (IL)-4 (FIG. 8A) and IL-5 (FIG. 8B) when re-stimulated exvivo by OVA.

FIG. 9 shows that there are more regulatory lymphocytes within lungtissue of Akkermansia muciniphila fed animals.

FIG. 10 is a graph illustrating the ability of Akkermansia muciniphilato induce LILRB2 (ILT4) expression by human dendritic cells.

FIG. 11 is a graph illustrating the ability of Akkermansia muciniphilato induce IL-10 secretion in a culture of human peripheral bloodmonocytes after generation of dendritic cells by exposure to GM-CSF andIL-4 for 6 days and contact with Akkermansia muciniphilia for 24 hours.

DETAILED DESCRIPTION

As indicated above, the present invention provides a compositioncomprising one or more Akkermansia muciniphila strains for use in theprophylaxis and/or treatment of undesirable inflammatory activitywherein said inflammatory activity is not caused by a metabolic disorderand/or obesity.

Of particular interest is that the invention is applicable toundesirable inflammatory activity remote from the gastrointestinaltract. Such undesirable inflammatory activity of concern may be airwayinflammatory activity such as that seen in the case of asthma includingexacerbations of asthma, COPD including exacerbations of COPD andeosinophilic COPD, infection-associated inflammation, allergen-inducedinflammation, allergic rhinitis or chronic rhinosinusitis. In anespecially preferred embodiment, the invention thus provides acomposition comprising one or more Akkermansia muciniphila strains foruse in the prophylaxis and/or treatment of airway inflammation ofasthma, especially in asthma patients with a lower than normal level ofAkkermansia muciniphilia in the gut. As indicated above, of particularinterest in relation to such use of Akkermansia muciniphilia are severeasthmatics. Asthma patients with a lower than normal gut level ofAkkermansia muciniphila may be conveniently pre-identified byapplication of various quantitative diagnostic tests to faecal samplesas further discussed below.

By way of example, it is envisaged that the invention may also findapplication for use in the prophylaxis and/or treatment of any of thefollowing: systemic inflammatory disease such as rheumatoid arthritis;undesirable inflammatory activity associated with an autoimmunedisorder; undesirable skin inflammatory activity such as associated withatopic dermatitis or psoriasis and inflammatory activity associated withan infection or cancer or immunotherapy thereof. It may be applied, forexample, for use in the prophylaxis and/or treatment of undesirableexacerbations of respiratory inflammatory activity due to bacterial orviral infections.

While use of the invention in relation to undesirable upper or lowerrespiratory inflammatory activity represents an embodiment of especialinterest, its use in relation to undesirable gastrointestinalinflammatory disorders is not excluded, for example use in theprophylaxis and/or treatment of undesirable gastrointestinalinflammatory activity as observed with, for example, inflammatory boweldisease such as Crohn's disease or ulcerative colitis, irritable bowelsyndrome, pouchitis, post-infection colitis or inflammation associatedwith gastrointestinal cancer. It may also find use in relation todiarrhoeal disease due to undesirable inflammatory activity, e.g. theprophylaxis and/or treatment of diarrhoeal disease due to an infectiousbacterial agent such as E. coli, Clostridium difficile associateddiarrhoea, Rotavirus associated diarrhoea or post-infective diarrhoea.

The one or more Akkermansia muciniphila strains employed may benaturally-occurring strains derived from human faeces or biopsies, ornaturally-occurring variants thereof, which have the desiredimmunomodulatory effect in the human gut. Genetically modified mutantsof naturally-occurring Akkermansia muciniphila strains may also beemployed provided they retain the desired activity. This may for examplebe equated with ability to induce ILT4 expression in human dendriticcells in vitro. A suitable assay for this purpose is set out in theexemplification below.

A composition for use in accordance with the invention may additionallyinclude one or more probiotic strains of another bacterial species suchas a Lactobacillus or Bifidobacterium, for example one or moreAkkermansia muciniphila strains may be combined with Bifidobacteriumlongum infantis 35624 or a variant or mutant thereof as described inEP1141235. However, any other probiotic bacterial strain withimmunological benefit might be considered. Alternatively the cells of adifferent species may be administered sequentially or simultaneously ina different composition.

A composition for use in accordance with the invention may be for directdelivery of one more Akkermansia muciniphila strains into the GI tract.Preferably a composition for use in accordance with the invention willbe a probiotic composition suitable for direct oral consumption.However, as indicated above a strain-release composition may beprovided, which is not ingested but is contacted with a liquid wherebysaid one or more strains are released into said liquid and the resultingprobiotic liquid composition is then consumed. For example, it may befavourable, particularly where the one or more probiotic strains are tobe delivered to children, to initially provide the one or more strainsin a composition, e.g. an oil suspension, within a drinking strawwhereby the one or more strains are transferred to a liquid drawnthrough the straw, e.g. a juice or milk from a drink carton. Such strawshousing probiotic strains are described, for example, in European Patentno. 1224128 (Biogaia AB).

A probiotic composition for oral administration in accordance with theinvention may include one or more Akkermansia muciniphila strains and aningestible carrier. It may preferably be in the form of a beverage, e.g.drinking yoghurt, juice or milk, or other form of nutritionalcomposition. It may be suitable for human and/or animal consumption. Forexample the composition may be in the form of a food product such as ayoghurt, cheese, confectionary, cereal or nutritional snack bar. It maybe in the form of a nutritional supplement. For example, the one or moreprobiotic strains may be provided in a nutritional oil suspension, e.g.within a capsule, which will release the probiotic strains in the GItract.

A composition for use in accordance with the invention may be formulatedas a pharmaceutical composition including a pharmaceutically acceptablecarrier, preferably such a formulation for oral administration includingan ingestible carrier. Such a composition may be in a solid form such asa tablet, pill, powder, granules, troches or suppository. It may be acontrolled-release formulation for release of the one or more probioticstrains in the GI tract. It may be in a liquid form, such as a liposomalcomposition or suspension, e.g. an oil suspension. Such a liquidcomposition may be provided within an encapsulating substance to providea capsule or microcapsules, again for release of the one or moreprobiotic strains in the GI tract. It may be a formulation for aerosoldelivery. A pharmaceutical composition for use in accordance with theinvention may additionally include a non-microbial therapeutic agent,e.g. a chemical drug entity or a therapeutic biologic, suitable for theprophylaxis and/or treatment of the undesired inflammatory activity ofconcern or an associated condition.

A composition for use in accordance with the invention will beformulated to provide an appropriate unit dosage of colony-forming units(cfu). This will vary with the form of administration and may be varied,for example, depending on whether the individual to be treated is achild or adult. For example a dosage of at least about 10⁴ cfu, morepreferably at least about 10⁵ cfu or at least about 10⁶ cfu, e.g. about10⁶-10¹⁰ cfu may be contemplated at least once per day. Administrationwill desirably be continued so that the level of Akkermansia muciniphilain the GI tract is at least restored to or maintained at around thenormal level to be expected for a healthy individual.

Akkermansia muciniphila, e.g. Akkermansia muciniphila Derrien et al(ATTC® BAA-835™) isolated from human faeces and obtainable from the ATCCas a freeze dried preparation, or Akkermansia muciniphilia similarlyisolated from human faeces or biopsies, may be grown anaerobically in amucin-based medium as previously described by Derrien et al. (11) andsubsequently incorporated into an appropriate composition foradministration as discussed above. For example, Akkermansia muciniphilathus cultured may be washed and at least initially suspended inanaerobic phosphate buffered saline (PBS), including 25% (v/v) glycerol,e.g. to an end concentration of 10¹⁰ cfu/ml under strict anaerobicconditions. Storage may be at −80° C. with subsequent thawing andpossible further dilution, e.g. with anaerobic PBS, as previouslydescribed (15, 16).

Due to the oxygen sensitivity of Akkermansia muciniphila, storage priorto use in accordance with the invention will be such as to protect fromcontact with air. Storage may be for example within lipid gels or drops.

Compositions for use in accordance with the invention will generally bestored prior to use in a sealed container or packaging to aid avoidanceof prolonged contact with air. Storage may be for example in the form ofa digestible solid composition, e g. pills, provided in sealedcompartments of a blister pack.

A composition for use in accordance with the invention by delivery tothe GI tract may additionally be supplemented with one or more prebioticsubstances. Such substances are recognized to promote growth of one ormore probiotic strains in the GI tract, but need not be metabolized by ahuman. The term ‘prebiotic’ will be understood to refer to anynon-viable food component that can be specifically fermented in thecolon by indigenous bacteria and which is considered of positive value.Types of probiotics of interest include non-digestible oligosaccharidesincluding those containing one or more of fructose, xylose, galactose,glucose and mannose. Soya may be employed as a prebiotic. The combinedadministration of a bacterial strain with one or more prebioticsubstances may enhance the growth of the strain in vivo resulting in amore pronounced health benefit, and is termed synbiotic.

It may be particularly preferred to employ a prebiotic substance whichwill promote growth in the GI tract of Akkermansia muciniphila. By wayof example, it has been shown that oligofructose will promote growth ofAkkermansia muciniphila in the GI tract of mice (16). It can beextrapolated that oligofructose, (e.g. a commercially availableoligofructose preparation such as an Orafti® oligofructose preparation,e.g. Orafti® P95 as supplied by Beneo GmbH) may be a useful component tobe administered as a prebiotic with one or more Akkermansia muciniphiliastrains in applying the invention. A prebiotic may be administered inthe same composition or separately, either simultaneously orsequentially.

In a further aspect of the invention, there is provided a prebioticsubstance, e.g. an oligofructose as discussed above, capable ofpromoting growth of Akkermansie muciniphila in the GI tract for use inthe prophylaxis and/or treatment of undesirable inflammatory activitywherein said inflammatory activity is not caused by a metabolic disorderand/or obesity. Such inflammatory activity of interest can be expectedto be accompanied by below normal level of Akkermansia muciniphila inthe GI tract and the prebiotic may be administered to raise orsubstantially maintain the level of Akkermansia muciniphila, preferablyso that it is restored to or maintained at or about the normal level fora healthy individual. The undesirable inflammatory activity may be anyundesirable inflammatory activity as discussed above, especially forexample undesirable airway inflammatory activity such as associated withasthma, COPD, eosinophilic COPD, infection-associated inflammation,allergen-induced inflammation, allergic rhinitis or chronicrhinosinusitis.

In an additional aspect of the invention, preferably compositionscontaining Akkermansia muciniphilia are employed as described above incombination with a diagnostic test to determine Akkermansia muciniphilaabundance in an individual with an aim of treating identifiedAkkermansia muciniphilia deficiency. Such a test may take the form offaecal bacterial microbiome 16S rRNA gene sequencing, PCR specificdetection of Akkermansia muciniphilia DNA or application of othernucleic acid hybridisation or sequencing technologies, or specificculture on selective media, for specific detection of Akkermansiamuciniphilia. Using the 16s sequencing data, the 75% percentile forrelative abundance of Akkermansia muciniphila in severe asthma patientsis 0.01422, which means that 1.422% of identifiable sequences areAkkermansia muciniphila. This suggests that Akkermansia muciniphilatreatment may be most effective in asthma patients whom have a relativeabundance of less than about 1.4%. Using the PCR method, the 75%percentile for Akkermansia muciniphila in severe asthma patients is 60.1copy numbering DNA, suggesting that Akkermansia muciniphila treatmentmay be most effective in asthma patients whom have a detected copynumber of less than about 60 per ng of total bacterial DNA.

The invention will be more clearly understood from the followingexamples.

EXAMPLES

It has been found, for the first time, that Akkermansia muciniphilalevels are significantly reduced in faecal samples from asthma patientsdisplaying partly controlled or uncontrolled asthma symptoms.Furthermore, consumption of Akkermansia muciniphila has been found tosignificantly reduce recruitment of disease-causing cells to the lungsin four different murine models of respiratory inflammation. Inaddition, Akkermansia muciniphila has immunomodulatory effects; it canmodulate cytokine secretion by human dendritic cells in vitro. Indeed,stimulation of the inhibitory molecule LILRB2 (ILT4) by Akkermansiamuciniphila is surprisingly significantly higher than that observed forother commensal microbes typically used as probiotics.

Four murine models for respiratory inflammation were employed to look atboth primarily neutrophil and eosinophil driven responses.

Neutrophils are a type of phagocyte and are normally found in thebloodstream. During the beginning (acute) phase of inflammation,particularly as a result of bacterial infection, environmental exposure,and some cancers, neutrophils are one of the first-responders ofinflammatory cells to migrate towards the site of inflammation. Theymigrate through the blood vessels, then through interstitial tissue,following chemical signals in a process called chemotaxis. They are thepredominant cells in pus, accounting for its whitish/yellowishappearance. Neutrophils release a wide range of proteins, which help todestroy a pathogen, but also have detrimental effects on host cells athigher concentrations. Asthma and asthma exacerbations can be associatedwith high neutrophil or high eosinophil numbers within the lung, both ofwhich contribute to the disease.

Eosinophils are white blood cells and one of the immune systemcomponents responsible for combating parasites and certain infections.Along with mast cells, they also control mechanisms associated withallergy and asthma. The presence of eosinophils in the lung or skin isassociated with disease. Eosinophils persist in the circulation for 8-12hours, and can survive in tissue for an additional 8-12 days in theabsence of stimulation. Eosinophils are important mediators of allergicresponses and asthma pathogenesis and are associated with diseaseseverity. Following activation, eosinophils release a range of powerfulmolecules including cationic granule proteins, reactive oxygen species,lipid mediators, enzymes, growth factors and cytokines. Many of themediators released by eosinophils are toxic at high levels to hostcells.

Evidence from murine house dust mite (HDM) re-challenge modelexperiments presented here and from recent literature demonstrate thatit is possible to identify two distinct populations of eosinophils inlung tissue based on Siglec-F expression. A lung resident population ofeosinophils has been identified as Siglec-F^(int), while an inflammatorypopulation, infiltrating after HDM re-challenge, is identified asSiglecF^(high). Inflammatory eosinophils are IL-5-dependent and havebeen shown to facilitate skewing towards a Th2 response, while theresident population are IL-5-independent and are thought to have a moreregulatory function responsible for inhibiting pro-allergic functions ofdendritic cells (19, 20). Mesnil and colleagues demonstrated that it isalso possible to stratify patients with asthma from healthy individualsbased on the presence of resident and inflammatory eosinophilpopulations within the lung with asthmatics having a higher number ofthe inflammatory eosinophil population; hence there has been suggested arelationship between eosinophil phenotype and disease.

Assays described below measured IgE, ILT4, IL-4, IL5, IL-10 and Foxp3⁺regulatory T cells.

IL-4 is a IL5-kd monomer (129 amino acids) produced by Th2 cells,basophils, mast cells, and eosinophils. There are two types of IL-4receptors. Type I IL-4R binds only IL-4 and consists of 2 receptorchains: IL-4Rα (CD124) and the common γc (CD132). Type II IL-4R bindsIL-4 and IL-13 and consists of the IL-4Rα and the IL-13Rα1 chains. Apleiotropic cytokine, IL-4 promotes allergic conditions and theprotective immune response against helminths and other extracellularparasites. IL-4 is the major stimulus of Th2-cell development; it alsosuppresses Th1-cell development and induces IgE class-switching in Bcells. IL-4 increases the expression of class II MHC molecules on Bcells, upregulates B-cell receptors, increases expression of CD23,prolongs lifespans of T and B cells in culture, and mediates tissueadhesion and inflammation. IL-4 and IL-4Rα knockout mice have defects inTh2-cell differentiation and reduced serum levels of IgG1 and IgE.

IL-5 is mainly produced by CD4+ Th2 cells, activated eosinophils, mastcells, CD8+Tc2 cells, γδ T cells, NK cells, NKT cells, andCD4-ckit-CD3ε-IL-2Rα+ cells in Payer patches. Its receptor shares theβ-chain (CD131) with IL-3 and GM-CSF, IL-5 promotes proliferation,activation, differentiation, survival, and adhesion of eosinophils. Th2cells that secrete IL-5 recruit eosinophils and contribute to theinduction of airway hyper-reactivity in patients with asthma. Levels ofIL-5, Th2 cells, and eosinophils are increased in bronchoalveolar lavageand correlate with asthma severity. IL-5-deficient mice develop normallybut are resistant to induction of experimental asthma, display reducedexpulsion of Nippostrongyius brasiliensis, and have fewer IgA+ antibodycells in the lamina propria compared with control mice. Clinical trialstargeting IL-5 have produced mixed results, but patients with refractoryeosinophilic asthma were reported to have reduced numbers ofexacerbations and eosinophils in sputum and blood and increased qualityof life (21).

FOXP3 (forkhoad box P3) also known as scurfin is a protein involved inimmune system responses. A member of the FOX protein family, FOXP3 is amaster regulator (transcription factor) in the development and functionof regulatory T cells. Regulatory T cells prevent excessive immuneresponses, which could damage host tissues. In autoimmune disease, adeficiency of regulatory T cell activity can allow other autoimmunecells to attack the body's own tissues. Defects in regulatory T cellinduction and/or activity have been demonstrated in multiple allergy andasthma models.

Immunoglobulin-like transcript 4 (ILT4) can be expressed on dendriticcells and is bound by HLA-G. The engagement of ILT4 by HLA-G tetramersprevents the up-regulation of costimulatory molecule expression,inhibits dendritic cell maturation and promotes the differentiation ofanergic/suppressor CD4⁺ T cells. In addition, differentiation of thecritical immunoregulatory IL-10 secreting T cell subset, Tr1 cells, isdependent on the IL-10-dependent ILT4/HLA-G pathway (22). These Tr1cells play a central role in tolerance induction and contribute to thesuppression of aberrant inflammatory activity, such as that observed inthe inflamed lung of asthma patients.

IL-10 is an anti-inflammatory cytokine that is an important regulator ofseveral aspects of immune responses (23). The IL-10 gene maps to acytokine cluster that includes the genes IL-19, IL-20, IL-24, and IL-26on chromosome 1q31-32. IL-10 is produced mainly by monocytes, T cells(mainly Tr1 cells), B cells, NK cells, macrophages, and dendritic cells.IL-10 is secreted as a homodimer that consists of 2 subunits, each of178 amino acids with a molecular weight of approximately 18 kd. IL-10directly affects antigen-presenting cell functions by down-regulatingthe expression of MHC class II and costimulatory molecules on thesurface of macrophages and monocytes. IL-10 inhibits the expression ofmany proinflammatory cytokines, chemokines, and chemokine receptors andmediates allergen tolerance in allergen-specific immunotherapy and afterexposure to high doses of allergen. In addition to these indirecteffects, IL-10 directly affects T-cell activation by suppressing CD28,CD2, and signaling of the inducible T-cell costimulator via the tyrosinephosphatase SHP-1. In contrast with its inhibitory effects on T cells,IL-10 promotes survival, proliferation, and differentiation of human Bcells and increases the production of IgG4. Several mouse modelsdemonstrate the importance of IL-10 in regulation of the inflammatoryresponse. IL-10 knockout mice develop normal lymphocyte and antibodyresponses but have reduced growth, are anemic, and spontaneously developchronic colitis.

Presence of elevated Immunoglobulin E (IgE) levels in serum is regardedas an integral part of the inflammatory cascade observed in allergicdiseases such as asthma. Induction of allergen-specific IgE can bedetected in animal models of allergic pulmonary inflammation as well asin human asthmatics and is an indication of the initiation of therelevant cellular (T and B cell axis) and humoral mechanisms that drivedevelopment of allergy. Hence, modulation of the allergen-specific IgEresponse by therapeutic intervention points to modulation of themechanisms that fundamentally underpin development of allergy. Inaddition, the importance of elevated levels of IgE is demonstrated bythe fact that anti-IgE antibody therapies have been shown to beimportant in reducing severe asthmatic exacerbations (24).

Example 1: Abundance of Bacterial Species in Faeces from Asthma Patients

Freshly voided faecal samples were obtained from asthma patients andwere stored at −80 C until analysis. Upon thawing, DNA was extractedfrom the faecal samples and the 16s rRNA V4 region was amplified by PCR.These PCR products were sequenced using MiSeq and identical sequenceswere grouped together into operational taxonomic units (OTUs). OTUs werealigned to known 16s sequences and the relative abundance for each OTUwas calculated.

In addition to microbiome analysis, clinical data was collected from allasthma patients. In particular, the level of symptom control wasassessed using an internationally validated questionnaire, which askedthe following questions.

Do you experience any of the following?

-   -   1. Asthma daytime symptoms more than 2 times per week?    -   2. Waking at night due to asthma symptoms?    -   3. Use of rescue medications more than 2 times in the previous        week?    -   4. Exercise or physical activity limited due to asthma symptoms?

A “No” answer to all four questions means that the asthma patient shouldbe considered as well controlled, A “Yes” answer to one or two questionsmeans that the patient is partly controlled, while a “Yes” answer tothree or four questions means that the patient has uncontrolled asthmasymptoms.

Surprisingly, when the microbiome data from asthma patients was dividedinto controlled, partly controlled and uncontrolled categories, it wasdiscovered that Akkermansia muciniphila levels were significantly lowerin patients with partly or uncontrolled asthma symptoms (FIG. 1A).Importantly, Akkermansia muciniphila levels in controlled asthmapatients were similar to that observed for healthy volunteers,representing approximately 8% of all the sequences obtained from theseindividuals. Thus, Akkermansia muciniphila is numerically one of themost dominant species within the colon. A reduction to approximately1-2% of the microbiome in patients with uncontrolled asthma symptoms isan enormous decrease with potentially critical health consequences. Asnoted above, this is the first time that a relationship betweendecreased gastrointestinal levels of Akkermansia muciniphila has beenlinked to the seventy of a respiratory disease, regardless of thepresence of obesity or atopy.

In order to confirm the 16s sequencing data, Akkermansia muciniphilalevels in faecal samples were also quantified using PCR. QuantitativePCR analysis was performed using an Applied Biosystems 7900 HT FastReal-Time PCR system and the Akkermansia muciniphila-specific primersequences used were AM1 5′CAG CAC GTG AAG GTG3′ (SEQ. ID. No, 1) and AM25′CCT TGC GGT TGG CTT CAG3′ (SEQ. ID. No, 2), The experimental cyclingconditions were: 40 cycles of 50° C. for 2 min, 95° C. for 10 min, 95°C. for 15 sec, 60° C. for 1 min. The quantitative PCR results wereidentical to the 16s sequencing data (FIG. 2).

Example 2. Akkermansia Muciniphila is Protective in the Murine Models ofAsthma

In order to understand if Akkermansia muciniphila has the potential todirectly influence lung inflammatory responses, we performed a murinehouse dust mite respiratory inflammation study. House dust mite (HDM)allergens are one of the most common aeroallergens that asthma patientsare allergic to. Intranasal (i.n.) administration of HDM extract to miceresults in lung inflammatory responses. We administered HDM extract i.n.on day 0 (1 μg), followed by higher dose i.n, administrations on days 7,8, 9, 10 and 11 (10 μg each day). This model is considered to be anacute challenge model. Animals were euthanized on day 14 andbronchoalveolar lavages (BAL) were obtained for enumeration ofinflammatory cells. Akkermansia muciniphila (from a stock culture of theinventors) was administered daily (approximately 1×10⁸ cells) by oralgavage, beginning at day −5 until the end of the study. Akkermansiamuciniphila was grown for 16 hours in 10 mL aliquots of anaerobic Mucinv3 media (10% inoculation) at 37° C. Nitrogen (and boiling) was used toremove the presence of oxygen from Mucin v3 media. Mucin v3 mediacontains peptone, yeast extract, KH₂PO₄, NaCl, (NH₄)SO₄, MgSO₄, CaCl₂,NaHCO₃, glucose, mucin type II, herein, cysteine and water, Negativecontrol animals received NaCl i.n. on the same days as the positivecontrol animals received HDM extracts i.n.

Surprisingly, oral administration of Akkermansia muciniphilasignificantly protected against inflammatory cell recruitment into theBAL of animals exposed to HDM extract, compared to animals that receivedHDM extract alone (FIG. 3A). When microscopic differential cell countswere performed, it was clear that the reduced BAL inflammatory cellcount in Akkermansia muciniphila treated animals was primarily due to areduced migration of neutrophils, which is the dominant infiltratingproinflammatory cell type in this model (FIG. 3B).

Chronic and Re-Challenge Models

Chronic intranasal challenges with proteolytically active allergens suchas house dust mite (HDM) extract have been shown to elicit allergicinflammatory changes within the lung (25). HDM extract is composed ofmany different substances that include proteolytic enzymes (Derp) andLPS and can therefore cause inflammatory responses that involve a vastarray of interactions between structural and immune cells (lymphocyticand myeloid) and mediators (26-28). In the experiments now presented,female BALB/c mice (8-10 weeks of age) were intra-nasally challengedwith either sterile saline or 25 μg of HDM extract in a volume of 50 μl(Greer labs, US, batch number: 218862) 5 days a week, for 3 weeks(sensitization period, days 1-19). This model is different to thatdescribed above and is considered to be a chronic model of respiratoryinflammation.

The resulting pulmonary inflammation in the HDM sensitized mice wasallowed to partially resolve for a period of 2 weeks (days 20 to 33). Atthe end of the resolution period (day 34), mice were intra-nasallychallenged with either saline or re-challenged with HDM (100 μg) in atotal volume of 50 μl. HDM re-challenge in this model has been shown toresult in granulocytic and lymphocytic infiltration into the lung over a7 day period (days 34 to 40). The pulmonary eosinophilic response in HDMre-challenged mice is significantly different to that of salinechallenged animals from 24 hrs (day 35) and peaks at 168 hrs (day 40)post HDM re-challenge. In the experiments presented here, mice weredosed by oral gavage (0.2 ml) with either media control or Akkermansiamuciniphila once a day, starting on day 20 (post cessation of HDMsensitization). Oral dosing continued throughout the resolution periodand post HDM re-challenge and finally ceasing on day 39. Groups of micewere sacrificed at pre-determined time-points during the resolutionphase of the experiment (day 28) and post HDM re-challenge (4 hrs (day34), 24 hrs (day 35) and 168 hrs (day 40), Cells present in thebroncho-alveolar lavage fluid (BALF) and lung tissue were analyzed andquantified using multi-colour flow cytometry.

Akkermansia muciniphila feeding resulted in enhanced clearance ofinflammatory cells (granulocytic and lymphocytic) present in the BALFduring the resolution phase of the experiment (FIGS. 4A and 4B).Infiltration of eosinophils into the BALF after HDM re-challenge wasalso significantly suppressed by Akkermansia muciniphila feeding (FIG.5A). Numbers of infiltrating inflammatory eosinophils (Siglec-F^(high)eos) were also significantly inhibited in the lung tissue of mice fedwith Akkermansia (FIGS. 5B and C). Akkermansia muciniphila was also ableto significantly inhibit the induction of HDM-specific IgE in the serumat day 40 (168 hrs post HDM re-challenge (FIG. 6)).

The HDM mouse model examines the response to an allergenic extract, theinventors also wished to test the influence of Akkermansia muciniphilain an eosinophil dominant model of lung disease, which is driven by asingle allergen. Mice were sensitized to the protein ovalbumin (OVA) byintraperitoneal injection (with the adjuvant alum) on days 0, 14 and 21followed by repeated OVA aerosol challenge on days 26-28. Animals wereeuthanized on day 29 for analysis of lung disease parameters.Akkermansia muciniphila purchased from the ATCC [Akkermansia muciniphilaDerrien et al. (ATTC® BAA-835™)] was administered by oral gavage(approximately 1×10⁸ cells) for the duration of the study. Akkermansiamuciniphila was again grown for 16 hours in 10 mL aliquots of anaerobicMucin v3 media (10% inoculation) at 37° C.

Surprisingly, Akkermansia muciniphila significantly protected againstinflammatory cell recruitment into the BAL of animals exposed to OVA,compared to animals that received OVA alone (FIG. 7A). When microscopicdifferential cell counts were performed, it was clear that the reducedBAL inflammatory cell count in Akkermansia muciniphila treated animalswas primarily due to a reduced migration of eosinophils, which is thedominant infiltrating proinflammatory cell type in this murine model(FIG. 7B).

Isolated cells from excised lung tissue were also re-stimulated with OVAin vitro to assess the cytokine response to OVA. Lymphocytes from OVAallergic animals secreted significantly more IL-4 and IL-5 in responseto OVA compared to lymphocytes from non-allergic animals (FIGS. 8A and8B), as expected. However, this increase in IL-4 and IL-5 secretion wasnot observed with lung tissue cells from animals treated withAkkermansia muciniphila by oral gavage.

Detailed analysis of the lung tissue lymphocytes revealed that there aresignificantly more Foxp3+ regulatory T cells that secrete IL-10 in thelung tissue of mice administered Akkermansia muciniphila (FIG. 9). Thesecells typically are potent suppressors of aberrant inflammatoryresponses. Thus increase in these cells within the lung may account forthe protective efficacy of Akkermansia muciniphila.

Example 3. Akkermansia muciniphila Induces Immune Regulatory andAnti-Inflammatory Responses in Dendritic Cells

A further study was carried out with a view to elucidating how thedramatic and unexpected anti-inflammatory lung effect could be inducedby a bacterium present within the gastrointestinal tract. Dendriticcells are one of the important cell types within the gut mucosa, whichsample luminal bacteria and induce potent polarizing effects ondownstream lymphocyte responses (29). Thus we hypothesized thatAkkermansia muciniphila stimulated dendritic cells to express highlevels of immune regulatory molecules, which would promote thedownstream anti-inflammatory effects observed in the lung.

Human monocyte-derived dendritic cells (MDDCs) were generated byculturing human peripheral blood monocytes in GM-CSF and IL-4 for sixdays. MDDCs were exposed to Akkermansia muciniphila for 24 hours andILT4 cell surface expression was measured by flow cytometry, while IL-10cytokine secretion was measured by RAGA. Akkermansia muciniphila wasprepared by growing for 16 hours in 10 mL aliquots of anaerobic Mucin v3media (10% inoculation) at 37° C. Bacterial cell number was determinedby microscopy and bacterial cells were added to MDDCs at a bacterialcell:MDDC cell ratio of 50:1.

Akkermansia muciniphila induced ILT4 expression on a very large numberof MDDCs (FIG. 10). Surprisingly, Akkermansia muciniphila induced ILT4expression was significantly higher than the effect observed with otherprobiotic microbes (such as Bifidobacteria or Lactobacilli) or forpathogens such as Staphylococcus aureus (FIG. 10), ILT4 is largelyresponsible for inducing regulatory T cells, which can secrete IL-10.Increased numbers of IL-10 positive regulatory T cells described in themurine model above may be due to induction of ILT4 expression bydendritic cells. In addition to inducing very high expression of theregulatory cell surface molecule ILT4, Akkermansia muciniphila alsoinduced very high levels of secretion, another anti-inflammatorymolecule (FIG. 11). Akkermansia muciniphila was prepared by growing for16 hours in 10 mL aliquots of anaerobic Mucin v3 media (10% inoculation)at 37° C. Bacterial cell number was determined by microscopy andbacterial cells were added to MDDCs at a bacterial cell:MDDC cell ratioof 10:1, 25:1 or 50:1.

This data suggests that the Akkermansia muciniphila protective effect inthe lung could be mediated in part via its induction of regulatorydendritic cells within the gut, which promote the local polarization ofregulatory lymphocytes and these regulatory lymphocytes can then migrateto other mucosal sites, such as the lung, to dampen proinflammatoryresponses.

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The invention claimed is:
 1. A method for the reducing inflammatory cellrecruitment in a subject having an airway disorder, wherein the airwaydisorder is asthma, exacerbations of asthma, infection-associatedinflammation, allergen-induced inflammation, allergic rhinitis orchronic rhinosinusitis, the method comprising administering one or moreAkkermansia muciniphilia strains to the gastrointestinal tract of asubject having an airway disorder and at risk of inflammatory cellrecruitment to the airway, thereby reducing inflammatory cellrecruitment in the subject.
 2. The method of claim 1, wherein saidairway disorder is asthma.
 3. The method of claim 1, wherein the subjecthas been identified as having a lower than normal level of Akkermansiamuciniphila in the gastrointestinal tract.
 4. The method of claim 2,wherein the subject has been identified as having a lower than normallevel of Akkermansia muciniphila in the gastrointestinal tract.
 5. Themethod of claim 3, wherein said subject has severe asthma.
 6. The methodof claim 1, wherein said one or more Akkermansia muciniphila strains areselected from a naturally-occurring Akkermansia muciniphila strainsderived from human feces or biopsies, or-naturally-occurring variantsthereof, which have an immunomodulatory effect in the human gut.
 7. Themethod of claim 1, wherein said one or more Akkermansia muciniphilastrains are in a probiotic composition suitable for oral administration.8. The method of claim 7, wherein said composition is a beverage orother form of nutritional composition.
 9. The method of claim 1, whereinsaid one or more Akkermansia muciniphila strains are in a pharmaceuticalcomposition suitable for delivery of said one or more strains to thegastrointestinal tract.
 10. The method of claim 1, wherein said one ormore Akkermansia muciniphila strains are formulated in a compositionwhich is packaged for aerosol delivery.
 11. The method of claim 1,wherein said one or more Akkermansia muciniphila strains areadministered simultaneously or sequentially with a prebiotic substancesuitable for promotion of growth of Akkermansia muciniphila in thegastrointestinal tract.
 12. The method of claim 1, wherein said one ormore Akkermansia muciniphila strains are present in a strain-releasecomposition and said method further comprises the pre-step of contactingsaid composition with a liquid, whereby said one or more strains arereleased into said liquid and the resulting probiotic liquid compositionis ingested.
 13. A method for preventing an episode of inflammatory cellrecruitment in a subject having an airway disorder, the methodcomprising administration of one or more Akkermansia muciniphiliastrains to the gastrointestinal tract of a subject having an airwaydisorder and at risk of an episode of inflammatory cell recruitment,thereby reducing the episode of inflammatory cell recruitment in thesubject.
 14. The method of claim 13, wherein the airway disorder isasthma, exacerbations of asthma, infection-associated inflammation,allergen-induced inflammation, allergic rhinitis or chronicrhinosinusitis.